Abstract The Polycomb Repressive Complex 2 (PRC2) is a highly conserved developmental regulator that maintains cellular identity by dynamically silencing key genes involved in differentiation. Alterations in EZH2, the catalytic methyltransferase, have been shown to play a driving role in many cancers. Activating mutations in EZH2 have been detected in a subset of cancers, such as melanoma and lymphomas. However, in most solid tumors, EZH2 is more commonly overexpressed. EZH2 expression levels progressively increase in advanced tumors, and has been functionally shown to drive prostate cancer metastasis. Nevertheless, the role of EZH2 in other solid tumors, including colorectal cancers (CRC) has not been sufficiently explored. Specifically, EZH2 is overexpressed in 78.5% of CRC, and its expression appears to inversely correlate with patient survival. We hypothesized that EZH2 could be an attractive therapeutic target, although its role and targets in CRC are unknown. CRC is the is one of the leading causes of cancer deaths worldwide, and advanced metastatic disease is still incurable. Thus, there is a significant unmet clinical need for treatments for CRC, especially those with activating mutations in KRAS. Many drugs that target classic oncogenic kinases are ineffective therapies as single agents, such as MEK inhibitors for KRAS mutant solid tumors. Therefore, one approach has been to develop more effective combination therapies that might enhance the sensitivity of cells to MEK inhibitors and/or prevent resistance. In a series of studies, our lab began exploring EZH2 inhibitor-based combination therapies in a variety of solid tumors. Interestingly, we have found that EZH2 inhibitors are frequently effective when combined with agents that target other key oncogenic pathways in a given tumor type, such as in breast and prostate cancer. We hypothesize that co-targeting EZH2 along with key oncogenic pathways may lead to cooperative killing of CRC cells by clamping down on crucial oncogenic signals at both the kinase level and the transcriptional level. My preliminary data demonstrate that a combination of EZH2 and MEK inhibitors cooperate to kill KRAS mutant CRC, which reveal a novel approach for treating this advanced disease. I propose to address several essential open-ended questions before translating a combination therapy into the clinic, such as the mechanism of action, the cell intrinsic factors that render tumors responsive to treatment, and whether the drug combination works in an in vivo preclinical and biologically relevant setting. In Aim 1, I propose to identify putative biomarkers that dictate sensitivity to EZH2/MEK inhibitors by conducting genomic analyses between sensitive and resistant cell lines. In Aim 2, I will elucidate the mechanism of action by obtaining a global view of chromatin and gene regulation, and I will identify critical PRC2 target genes that modulated in CRC after combination treatment. In Aim 3, I will test the eff...